Abstract Chromosomal translocations of the gene coding for the epigenetic signaling protein MLL1 are frequent events in acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). These MLL1 fusion leukemias are consistently poor prognosis, highlighting the need for novel approaches to treatment. We have shown that the CXXC domain of MLL1, which is retained in the leukemia driving fusion proteins, binds specifically to nonmethylated CpG elements and protects them from methylation. Furthermore, introduction of point mutations into the MLL1 CXXC domain which disrupt DNA binding in the context of an MLL-AF9 fusion protein completely abrogates the ability of the fusion protein to cause leukemia in vivo. This data validates the MLL1 CXXC domain as a target for drug development for the treatment of MLL fusion leukemia. We have developed fluorescence polarization assays for the binding of the MLL1 CXXC domain to DNA. We screened two fragment libraries using this assay and confirmed 36 hits by chemical shift perturbations in 15N-1H HSQC spectra of the CXXC domain plus compounds. Our structural data showed there is one solvent exposed Cys residue located at the DNA binding interface. Based on this, we have also screened a library of Cys reactive molecules and identified 6 hits. We are proposing to covalently link an active fragment with a compound derived from the Cys reactive library which bind to separate sites on the protein to generate a potent inhibitor of the MLL1 CXXC domain. Once an effective inhibitor is generated, we will profile its effects on the growth of MLL1 fusion leukemia cell lines versus non-MLL1 fusion leukemia cell lines to establish selectivity of action. Furthermore, we will assess effects on DNA methylation and gene expression of well-characterized target genes. Based on this, we are proposing 2 aims: Aim 1: Development of a potent and specific inhibitor of the MLL1 CXXC domain. We will develop a potent and specific inhibitor of the MLL1 CXXC domain by covalently linking a fragment and a compound derived from the Cys reactive library which bind to separate sites on the protein. Aim 2: Validation of on-target activity of MLL1 CXXC domain inhibitor. We will validate the MLL1 CXXC domain inhibitor by testing effects on MLL fusion leukemia cell line proliferation, DNA methylation at target genes, and gene expression at target genes.